7. PROJECT SUMMARY/ABSTRACT The mitochondrial inner membrane is the site of essential cellular functions such as oxidative phosphorylation, phospholipid metabolism, and the regulation of apoptosis. The inner membrane is under constant assault from reactive oxygen species, inevitable by-products of respiration. To limit the effects of this damage and to maintain proteostasis throughout mitochondria, AAA+ proteases harness the energy of ATP to recognize, unfold and degrade protein substrates both from within and surrounding the inner membrane. AAA+ proteases assemble as hexamers to form an internal proteolytic chamber into which substrates are forcibly translocated by an ATPase module. The ATPase active site is created by interactions from adjacent subunits such that oligomerization is a requirement for activity. The mitochondrial AAA+ proteases YME1L and AFG3L2 are largely soluble enzymes that are anchored in the inner membrane and represent a significantly understudied class of proteolytic system that operate at the membrane interface. In humans, dysfunction of these proteases has been linked to the development of severe neurodegenerative disorders such as spinocerebellar ataxia. Understanding the molecular mechanisms of these important enzymes has been hampered by the difficulty in studying membrane-anchored enzymes in vitro. I have developed a novel approach to assemble previously membrane-constrained hexameric proteases in a soluble, active form. This breakthrough allows for the application of established solution biochemical and biophysical techniques to the study of proteostasis at the mitochondrial inner membrane for the first time. The first aim of the proposal is to define how substrate proteins are selected for degradation among the myriad proteins housed in the inner membrane. Both model proteins and known physiological substrates will be used to determine what features are necessary and sufficient to drive degradation. The second aim is analyze the coordination of ATP hydrolysis and the production of pulling forces to understand how these molecular machines are capable of extracting substrates from within the inner membrane. Finally, the ability to produce large quantities of soluble active protease will be leveraged to determine crystal structures of the proteases in their active state, and in complex with nucleotide and protein substrates. Together, these experiments will form the first rigorous mechanistic analysis of the mitochondrial AAA+ proteases and provide foundational knowledge to aid the development of small molecule modulators as future therapeutics.